This project involves a study of the regulation of histidine and threonine catabolism in Pseudomonas putida by concentrating on the control properties of histidase, urocanase and threonine dehydratase. Threonine dehydratase of this organism is a dual function enzyme, involved in isoleucine biosynthesis as well as threonine catabolism. We are attempting to establish the existence of a covalently modified form of the enzyme by examination of enzymic properties and by mutant construction and analysis. We propose that the covalent modification is regulated by nitrogen availability and is employed by the cell as a means of controlling isoleucine biosynthesis. Related studies propose an examination of the roles of cyclic AMP and glutamine synthetase in favoring transcription of genes for histidase, urocanase and the other hut enzymes. Mutants altered in cyclic AMP production of glutamine synthetase activity are being isolated. The allosteric regulation of histidase is also being examined. Further studies are anticipated on the NAD-containing urocanase, with the aim being to elucidate mechanistic features by isotope exchange and NMR analysis. NAD binding measurements will also be made. Purification of beef liver urocanase will also be attempted to permit generalization of NAD as coenzyme for other urocanases.